Bottom Line:
Seventeen conserved and 226 novel miRNAs differed at least 2-fold in response to blue and UV-A light compared with levels after a dark treatment.Real time PCR showed that BrmiR159, BrmiRC0191, BrmiRC0460, BrmiRC0323, BrmiRC0418, BrmiRC0005 were blue light-induced and northern blot revealed that the transcription level of BrmiR167 did not differ significantly among seedlings treated with dark, blue or UV-light.A set of miRNAs and their targets involved in the regulation of the light-induced photomorphogenic phenotype in seedlings of Brassica rapa was identified, providing new insights into blue and UV-A light-responsive miRNAs in seedlings of Tsuda and evidence of multiple targets for the miRNAs and their diverse roles in plant development.

Background: Growth, development, and pigment synthesis in Brassica rapa subsp. rapa cv. Tsuda, a popular vegetable crop, are influenced by light. Although microRNAs (miRNAs) have vital roles in the metabolic processes and abiotic stress responses of plants, whether miRNAs play a role in anthocyanin biosynthesis and development of Tsuda seedlings exposed to light is unknown.

Results: Seventeen conserved and 226 novel miRNAs differed at least 2-fold in response to blue and UV-A light compared with levels after a dark treatment. Real time PCR showed that BrmiR159, BrmiRC0191, BrmiRC0460, BrmiRC0323, BrmiRC0418, BrmiRC0005 were blue light-induced and northern blot revealed that the transcription level of BrmiR167 did not differ significantly among seedlings treated with dark, blue or UV-light. BrmiR156 and BrmiR157 were present in the greatest amount (number of reads) and among their 8 putative targets in the SPL gene family, only SPL9 (Bra004674) and SPL15 (Bra003305) increased in expression after blue or UV-A exposure. In addition, miR157-guided cleavage of target SPL9 mRNAs (Bra004674, Bra016891) and SPL15 mRNAs (Bra003305, Bra014599) took place 10 or 11 bases from the 5' ends of the binding region in the miR157 sequence.

Conclusions: A set of miRNAs and their targets involved in the regulation of the light-induced photomorphogenic phenotype in seedlings of Brassica rapa was identified, providing new insights into blue and UV-A light-responsive miRNAs in seedlings of Tsuda and evidence of multiple targets for the miRNAs and their diverse roles in plant development.

Fig4: Northern blot analysis of BrmiR156, BrmiR167 and BrmiR159 in seedlings of Tsuda after exposure to dark, blue light or UV-A. 5′-Digoxigenin-labeled DNA oligonucleotide with complementary sequence to miRNA was used as the probe; BrU6 was used as the control. The gray level ratio between miRNAs and BrU6 was used to compare relative expression to normalized reads

Mentions:
To confirm the original high-throughput sequencing results, we selected three conserved miRNAs (BrmiR156 with more than 3 million reads, BrmiR167 with more than 100,000 reads, and BrmiR159 with more than 2000 reads) as examples to analyze their expression using an RNA blot. In addition, five novel candidate miRNAs and BrmiR159 were analyzed by real time PCR. The RNA blot and real time PCR results demonstrated that all tested miRNAs were expressed in seedlings in the dark, blue light and UV-A treatments and that the transcription levels of the different miRNAs varied (Figs. 3, 4). Transcription of most of the selected miRNAs (BrmiR159, BrmiRC0191, BrmiRC0460, BrmiRC0323, BrmiRC0418, BrmiRC0005) was higher after the blue light treatment than after the dark or the UV-A light treatment (Fig. 3). Northern blot showed that the transcription level of BrmiR167 was not significantly different among the seedlings from the dark, blue and UV-light induction, while the expression of BrmiR156 was slightly inhibited by blue and UV-A light (Fig. 4). When the relative expression levels of candidate microRNAs shown in RNA blots (gray level ratios between the blot results of microRNAs and the U6) or by RT-PCR were compared with the normalized reads of microRNAs from the high-throughput sequencing library of the dark, blue light and UV-A light induced seedlings, all selected miRNAs had the same expression profiles as in the original high-throughput sequencing results (Fig. 3, 4).Fig. 3

Fig4: Northern blot analysis of BrmiR156, BrmiR167 and BrmiR159 in seedlings of Tsuda after exposure to dark, blue light or UV-A. 5′-Digoxigenin-labeled DNA oligonucleotide with complementary sequence to miRNA was used as the probe; BrU6 was used as the control. The gray level ratio between miRNAs and BrU6 was used to compare relative expression to normalized reads

Mentions:
To confirm the original high-throughput sequencing results, we selected three conserved miRNAs (BrmiR156 with more than 3 million reads, BrmiR167 with more than 100,000 reads, and BrmiR159 with more than 2000 reads) as examples to analyze their expression using an RNA blot. In addition, five novel candidate miRNAs and BrmiR159 were analyzed by real time PCR. The RNA blot and real time PCR results demonstrated that all tested miRNAs were expressed in seedlings in the dark, blue light and UV-A treatments and that the transcription levels of the different miRNAs varied (Figs. 3, 4). Transcription of most of the selected miRNAs (BrmiR159, BrmiRC0191, BrmiRC0460, BrmiRC0323, BrmiRC0418, BrmiRC0005) was higher after the blue light treatment than after the dark or the UV-A light treatment (Fig. 3). Northern blot showed that the transcription level of BrmiR167 was not significantly different among the seedlings from the dark, blue and UV-light induction, while the expression of BrmiR156 was slightly inhibited by blue and UV-A light (Fig. 4). When the relative expression levels of candidate microRNAs shown in RNA blots (gray level ratios between the blot results of microRNAs and the U6) or by RT-PCR were compared with the normalized reads of microRNAs from the high-throughput sequencing library of the dark, blue light and UV-A light induced seedlings, all selected miRNAs had the same expression profiles as in the original high-throughput sequencing results (Fig. 3, 4).Fig. 3

Bottom Line:
Seventeen conserved and 226 novel miRNAs differed at least 2-fold in response to blue and UV-A light compared with levels after a dark treatment.Real time PCR showed that BrmiR159, BrmiRC0191, BrmiRC0460, BrmiRC0323, BrmiRC0418, BrmiRC0005 were blue light-induced and northern blot revealed that the transcription level of BrmiR167 did not differ significantly among seedlings treated with dark, blue or UV-light.A set of miRNAs and their targets involved in the regulation of the light-induced photomorphogenic phenotype in seedlings of Brassica rapa was identified, providing new insights into blue and UV-A light-responsive miRNAs in seedlings of Tsuda and evidence of multiple targets for the miRNAs and their diverse roles in plant development.

Background: Growth, development, and pigment synthesis in Brassica rapa subsp. rapa cv. Tsuda, a popular vegetable crop, are influenced by light. Although microRNAs (miRNAs) have vital roles in the metabolic processes and abiotic stress responses of plants, whether miRNAs play a role in anthocyanin biosynthesis and development of Tsuda seedlings exposed to light is unknown.

Results: Seventeen conserved and 226 novel miRNAs differed at least 2-fold in response to blue and UV-A light compared with levels after a dark treatment. Real time PCR showed that BrmiR159, BrmiRC0191, BrmiRC0460, BrmiRC0323, BrmiRC0418, BrmiRC0005 were blue light-induced and northern blot revealed that the transcription level of BrmiR167 did not differ significantly among seedlings treated with dark, blue or UV-light. BrmiR156 and BrmiR157 were present in the greatest amount (number of reads) and among their 8 putative targets in the SPL gene family, only SPL9 (Bra004674) and SPL15 (Bra003305) increased in expression after blue or UV-A exposure. In addition, miR157-guided cleavage of target SPL9 mRNAs (Bra004674, Bra016891) and SPL15 mRNAs (Bra003305, Bra014599) took place 10 or 11 bases from the 5' ends of the binding region in the miR157 sequence.

Conclusions: A set of miRNAs and their targets involved in the regulation of the light-induced photomorphogenic phenotype in seedlings of Brassica rapa was identified, providing new insights into blue and UV-A light-responsive miRNAs in seedlings of Tsuda and evidence of multiple targets for the miRNAs and their diverse roles in plant development.